A brief discussion of the volume-scale of chromatography and its effect on fluid-path implementation is given below. High-performance liquid chromatography (HPLC) is traditionally performed using analytical columns having a finished internal diameter (ID) or bore of about 4.6 millimeters, and a length typically in the range of about 5 centimeters to 25 centimeters. Such columns are typically assembled from carefully machined components.
The column tube typically has a male thread on each end, which engages a corresponding female thread within respective column end-fittings or terminals. Each column end-fitting incorporates features critical to the performance of the finished column. Among these features is a fit and diffuser plate, which cooperate to retain the particulate stationary phase (or packing) within the column bed, and to transition the liquid flow between the geometry of the narrow-bore input/output interconnect tubing (typically 0.23 millimeters ID) and the much broader diametral dimension of the packed bed (4.6 millimeters.) Each column end-fitting also includes a threaded compression port, which is used to establish a substantially leak-tight interface between the column and an interconnect tube.
A traditional 4.6 mm ID HPLC column might be packed with a stationary phase incorporating a characteristic packing-particle diameter of 5 micrometers. Operation of that column at a suitable mobile-phase volumetric flow rate will result in a characteristic mobile-phase linear velocity through the bed structure. If the column is well-packed (i.e. in the substantial absence of voids, bridges, or other bed defects) then this operating regime will result in a characteristic separation “efficiency” for this system, as demonstrated through the use of one or more types of probe compounds. The characteristic efficiency may be thought of as a measure of the narrowness of the chromatographic zones or bands which may be propagated through the system.
In an HPLC analytical instrument, it is generally desirable to perform separations with high efficiency, thereby maximizing information content of the chromatogram by enhancing the resolution of interfering or near-interfering zones or bands. A band which is eluted from the above-described system might be expected to have a time-course of substantially 10 seconds, measured at 5-sigma (i.e., passage of the substantially Gaussian concentration-distribution of the band through a detector, including the band apex, as well as 2.5-sigma of the band preceding and trailing the apex.)
With knowledge of the volumetric flow rate, one can convert the width of the band in time units to a width in volume units (167 microliters in this example, for a flow rate of 1.0 milliliter per minute. Working in the volume domain is particularly instructive as one proceeds to investigate the impact of “extra-column” volumes on the efficiency of the separation. The existence of volumes external to the column (for example, in transport tubing, in detectors, and in injectors) generally can only degrade the quality of a separation as delivered by a column.
The extra-column variance (variance=sigma2) contribution is an extremely useful measure to illuminate how a specified separation will be degraded in the presence of one or more types of extra-column contributions, as variances are substantially additive. It is instructive to tabulate the characteristic volume scale of several classes of chromatography systems, to perceive what the system designer is confronted with. In the tabulation below, the assumption is made that all systems will preserve the same efficiency value, and that mobile-phase linear velocity will be maintained constant through the packed bed. Thus, the volumetric flow rate has been scaled in proportion to column bed cross-sectional area, thus in proportion to column internal radius2.
TABLE 1CharacteristicHPLC ScaleColumn IDVolumetric Flow RatePeak VolumeConv. Analytical3.9-4.6mm1.0milliliter/min167-200microlitersNarrow-bore2.0mm250microliters/min40-50microlitersMicrobore1.0mm50-70microliters/min10-12microlitersCapillary0.30-0.50mm5-12microliters/min1.5-2.5microlitersNanoscale0.05-0.15mm10's-100'snanoliters/min10-40nanoliters
It will be further recognized that as one transitions to the use of smaller appropriately-packed particles to construct the chromatography column bed, one can achieve higher separation efficiencies. This regime, as commercialized by Waters Corporation (Milford, Mass., USA) using nominal 1.7 micrometer diameter particles, has been termed Ultra Performance Liquid Chromatography™ (UPLC™). The bands or zones in UPLC™ are thus narrower (in both the time domain and the volume domain) than their counterparts in HPLC, placing further demands on reduction of extra-column volume and its zone-dispersion effects.
The column and flow-rate ranges of Table 1 illustrate how conventional tubing and tubing-interfaces, which are quite satisfactory for use in conventional-scale HPLC (where characteristic peak volumes are a significant fraction of a milliliter,) are quickly outclassed in applications such as capillary-scale or nanoscale HPLC (where characteristic peak volumes are in the few-microliter to tens-of-nanoliters range, and thus the extra-column variance “budget” is essentially gone.) Stated another way, extra-column volumes and extra-column variances that are acceptable in the practice of conventional HPLC are generally inappropriate in the practice of capillary and nanoscale LC techniques. Capillary and nanoscale techniques are at the forefront of separations technology at this time, largely because of their suitability for interfacing with mass spectrometry, particularly where the available sample-mass for analysis is limited (sample-limited analysis.)
In practice, few if any manufacturers have demonstrated the ability to maintain separation efficiency across the orders-of-magnitude of characteristic peak volume recited in Table 1. Moreover, there is concurrently a trend toward the use of smaller packing particle size, to achieve yet-higher separation efficiency. This higher efficiency results in a further decrease in the volume of an eluting zone or band, further exacerbating problems with extra column effects. Planar fluid-circuit approaches to minimizing extra-column volume and extra-column variance seem appealing in their ability to consolidate function and produce relatively short routing paths, but to date the materials of construction (typically glass, plastics, or certain ceramics) have not permitted the devices to withstand the internal hydrostatic pressures typical of modern small-particle separations. These latter pressures may be in the range of 100 megapascals at the column head or higher, corresponding to regimes of UPLC™ or of very-high-pressure liquid chromatography (VHPLC.)